The present invention provides peptides and compounds that bind to and activate the thrombopoietin receptor (c-mpl or TPO-R) or otherwise act as a TPO agonist. The invention has application in the fields of biochemistry and medicinal chemistry and particularly provides TPO agonists for use in the treatment of human disease.
Megakaryocytes are bone marrow-derived cells, which are responsible for producing circulating blood platelets. Although comprising  less than 0.25% of the bone marrow cells in most species, they have  greater than 10 times the volume of typical marrow cells. See Kuter, et. al., Proc. Natl. Acad. Sci. USA 91:11104-11108 (1994). Megakaryocytes undergo a process known as endomitosis whereby they replicate their nuclei but fail to undergo cell division and thereby give rise to polyploid cells. In response to a decreased platelet count, the endomitotic rate increases, higher ploidy megakaryocytes are formed, and the number of megakaryocytes may increase up to 3-fold. See Harker, J. Clin. Invest., 47:458-465 (1968). In contrast, in response to an elevated platelet count, the endomitotic rate decreases, lower ploidy megakaryocytes are formed, and the number of megakaryocytes may decrease by 50%.
The exact physiological feedback mechanism by which the mass of circulating platelets regulates the endomitotic rate and number of bone marrow megakaryocytes is not known. The circulating thrombopoietic factor involved in mediating this feedback loop is now thought to be thrombopoietin (TPO). More specifically, TPO has been shown to be the main humoral regulator in situations involving thrombocytopenia. See, e.g., Metcalf, Nature, 369:519-520 (1994). TPO has been shown in several studies to increase platelet counts, increase platelet size, and increase isotope incorporation into platelets of recipient animals. Specifically, TPO is thought to affect megakaryocytopoiesis in several ways: (1) it produces increases in megakaryocyte size and number; (2) it produces an increase in DNA content, in the form of polyploidy, in megakaryocytes; (3) it increases megakaryocyte endomitosis; (4) it produces increased maturation of megakaryocytes; and (5) it produces an increase in the percentage of precursor cells, in the form of small acetylcholinesterase-positive cells, in the bone marrow.
Because platelets (thrombocytes) are necessary for blood clotting and when their numbers are very low a patient is at serious risk of death from catastrophic hemorrhage, TPO has potential useful application in both the diagnosis and the treatment of various hematological disorders, for example, diseases primarily due to platelet defects. Ongoing clinical trials with TPO have indicated that TPO can be administered safely to patients. In addition, recent studies have provided a basis for the projection of efficacy of TPO therapy in the treatment of thrombocytopenia, and particularly thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transplantation as treatment for cancer or lymphoma. See, e.g., McDonald, Am. J. Ped. Hematology/Oncology, 14:8-21 (1992).
The gene encoding TPO has been cloned and characterized. See Kuter, et al., Proc. Natl. Acad. Sci. USA, 91:11104-11108 (1994); Barley, et al., Cell 77:1117-1124 (1994); Kaushansky et al., Nature 369:568-571 (1994); Wendling, et al., Nature, 369:571-574 (1994); and Sauvage et al., Nature 369:533-538 (1994). Thrombopoietin is a glycoprotein with at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, with a common N-terminal amino acid sequence. See, Bartley, et al., Cell, 77:1117-1124 (1994). Thrombopoietin appears to have two distinct regions separated by a potential Arg-Arg cleavage site. The amino-terminal region is highly conserved in man and mouse, and has some homology with erythropoietin and interferon-a and interferon-b. The carboxy-terminal region shows wide species divergence.
The DNA sequences and encoded peptide sequences for human TPO-R (also known as c-mpl) have been described. See Vigon, et al., Proc. Natl. Acad. Sci. USA, 89:5640-5644 (1992). TPO-R is a member of the hematopoietin growth factor receptor family, a family characterized by a common structural design of the extracellular domain, including four conserved C residues in the N-terminal portion and a WSXWS motif close to the transmembrane region. See Bazan, Proc. Natl. Acad. Sci. USA, 87:6934-6938 (1990). Evidence that this receptor plays a functional role in hematopoiesis includes observations that its expression is restricted to spleen, bone marrow, or fetal liver in mice (see Souyri, et al., Cell 63:1137-1147 (1990)) and to megakaryocytes, platelets, and CD34+ cells in humans (see Methia, et al., Blood 82:1395-1401 (1993)). Furthermore, exposure of CD34+ cells to synthetic oligonucleotides antisense to mpl RNA significantly inhibits the appearance of megakaryocyte colonies without affecting erythroid or myeloid colony formation. Some workers postulate that the receptor functions as a homodimer, similar to the situation with the receptors for G-CSF and erythropoietin.
The availability of cloned genes for TPO-R facilitates the search for agonists of this important receptor. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the xe2x80x9cpeptides on plasmidsxe2x80x9d system described in U.S. Pat. Nos. 5,270,170 and 5,338,665; the xe2x80x9cpeptides on phagexe2x80x9d system described in U.S. patent application Ser. No. 07/718,577, filed Jun. 20, 1991, U.S. patent application Ser. No. 07/541,108, filed Jun. 20, 1990, and in Cwirla, et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990); the xe2x80x9cpolysomexe2x80x9d system described in U.S. patent application Ser. No. 08/300,262, filed Sep. 2, 1994, which is a continuation-in-part application based on U.S. patent application Ser. No. 08/144,775, filed Oct. 29, 1993 and PCT WO 95/11992; the xe2x80x9cencoded synthetic libraryxe2x80x9d system described in U.S. patent application Ser. Nos. 08/146,886, filed Nov. 12, 1993, 07/946,239, filed Sep. 16, 1992, and 07/762,522, filed Sep. 18, 1991; and the xe2x80x9cvery large scale immobilized polymer synthesisxe2x80x9d system described in U.S. Pat. No. 5,143,854; PCT Patent Publication No. 90/15070, published Dec. 13, 1990; U.S. patent application Ser. No. 07/624,120, filed Dec. 6, 1990; Fodor, et al., Science, 251:767-773 (2/1991); Dower, et al., Ann. Rep. Med. Chem., 26:271-180 (1991); and U.S. patent application Ser. No. 07/805,727, filed Dec. 6, 1991; each of the foregoing patent applications and publications is incorporated herein by reference.
The slow recovery of platelet levels in patients suffering from thrombocytopenia is a serious problem, and has lent urgency to the search for a blood growth factor agonist able to accelerate platelet regeneration. The present invention provides such an agonist.
This invention is directed, in part, to the novel and unexpected discovery that defined low molecular weight peptides and peptide mimetics have strong binding properties to the TPO-R and can activate the TPO-R. Accordingly, such peptides and peptide mimetics are useful for therapeutic purposes in treating conditions mediated by TPO (e.g., thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions) as well as for diagnostic purposes in studying the mechanism of hematopoiesis and for the in vitro expansion of megakaroycytes and committed progenitor cells.
Peptides and peptide mimetics suitable for therapeutic and/or diagnostic purposes have an IC50 of about 2 mM or less, as determined by the binding affinity assay set forth in Example 3 below wherein a lower IC50 correlates to a stronger binding affinity to TPO-R. For pharmaceutical purposes, the peptides and peptidomimetics preferably have an IC50 of no more than about 100 xcexcm, more preferably, no more than 500 nM. In a preferred embodiment, the molecular weight of the peptide or peptide mimetic is from about 250 to about 8,000 daltons. If the peptides of this invention are oligomerized, dimerized and/or derivatized with a hydrophilic polymer as described herein, the molecular weights of such peptides will be substantially greater and can range anywhere from about 500 to about 120,000 daltons, more preferable from about 8,000 to about 80,000 daltons.
When used for diagnostic purposes, the peptides and peptide mimetics preferably are labeled with a detectable label and, accordingly, the peptides and peptide mimetics without such a label serve as intermediates in the preparation of labeled peptides and peptide mimetics.
Peptides meeting the defined criteria for molecular weight and binding affinity for TPO-R comprise 9 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Peptide mimetics include peptides having one or more of the following modifications:
peptides wherein one or more of the peptidyl [xe2x80x94C(O)NRxe2x80x94] linkages (bonds) have been replaced by a non-peptidyl linkage such as a xe2x80x94CH2xe2x80x94 carbamate linkage [xe2x80x94CR2xe2x80x94OC(O)NRxe2x80x94]; a phosphonate linkage; a xe2x80x94CH2xe2x80x94sulfonamide [xe2x80x94CH2xe2x80x94S(xc2x0)2NRxe2x80x94] linkage; a urea [xe2x80x94NHC(O)NHxe2x80x94] linkage; a xe2x80x94CH2xe2x80x94 secondary amine linkage; or an alkylated peptidyl linkage [xe2x80x94C(O)NR6xe2x80x94 where R6 is lower alkyl];
peptides wherein the N-terminus is derivatized to a xe2x80x94NRR1 group; to a xe2x80x94NRC(O)R group; to a xe2x80x94NRC(O)OR group; to a xe2x80x94NRS(O)2R group; to a xe2x80x94NHC(O)NHR group where R and R1 are hydrogen or lower alkyl with the proviso that R and R1 are not both hydrogen; to a succinimide group; to a benzyloxycarbonyl-NHxe2x80x94 (CBZ-NHxe2x80x94) group; or to a benzyloxycarbonyl-NHxe2x80x94 group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo; or
peptides wherein the C terminus is derivatized to xe2x80x94C(O)R2 where 2 is selected from the group consisting of lower alkoxy, and xe2x80x94NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl.
Accordingly, preferred peptides and peptide mimetics comprise a compound having:
(1) a molecular weight of less than about 5000 daltons, and
(2) a binding affinity to TPO-R as expressed by an IC50 of no more than about 100 xcexcm,
wherein from zero to all of the xe2x80x94C(O)NHxe2x80x94 linkages of the peptide have been replaced by a linkage selected from the group consisting of a xe2x80x94CH2OC(O)NRxe2x80x94 linkage; a phosphonate linkage; a xe2x80x94CH2S(O)2NRxe2x80x94 linkage; a xe2x80x94CH2NRxe2x80x94 linkage; and a xe2x80x94C(O)NR6xe2x80x94linkage; and a xe2x80x94NHC(O)NHxe2x80x94 linkage where R is hydrogen or lower alkyl and R6 is lower alkyl,
further wherein the N-terminus of said peptide or peptide mimetic is selected from the group consisting of a xe2x80x94NRR1 group; a xe2x80x94NRC(O)R group; a xe2x80x94NRC(O)OR group; a xe2x80x94NRS(O)2R group; a xe2x80x94NHC(O)NHR group; a succinimide group; a benzyloxycarbonyl-NHxe2x80x94 group; and a benzyloxycarbonyl-NHxe2x80x94 group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo, where R and R1 are independently selected from the group consisting of hydrogen and lower alkyl, and still further wherein the C-terminus of said peptide or peptide mimetic has the formula xe2x80x94C(O)R2 where R2 is selected from the group consisting of hydroxy, lower alkoxy, and xe2x80x94NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl and where the nitrogen atom of the xe2x80x94NR3R4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide,
and physiologically acceptable salts thereof.
In a related embodiment, the invention is directed to a labeled peptide or peptide mimetic comprising a peptide or peptide mimetic described as above having covalently attached thereto a label capable of detection.
In some embodiments of the invention, preferred peptides for use include peptides having a core structure comprising a sequence of amino acids:
X1 X2 X3 X4 X5 X6 X7 
where X1 is C, L, M, P, Q, V; X2 is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, L, M, R, S, V or W; X4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V.
In a preferred embodiment the core peptide comprises a sequence of amino acids:
X8 G X1 X2 X3 X4X5 W X7 
where X1 is L, M, P, Q, or V; X2 is F, R, S, or T; X3 is F, L, V, or W; X4 is A, K, L, M, R, S, V, or T; X5 is A, E, G, K, M, Q, R, S, or T; X7 is C, I, K, L, M or V; and each X8 residue is independently selected from any of the 20 genetically coded L-amino acids, their stereoisomeric D-amino acids; and non-natural amino acids. Preferably, each X8 residue is independently selected from any of the 20 genetically coded L-amino acids and their stereoisomeric D-amino acids. In a preferred embodiment, X1 is P; X2 is T; X3 is L; X4 is R; X5 is E or Q; and X7 is I or L.
More preferably, the core peptide comprises a sequence of amino acids:
X9 X8 C X1 X2 X3 X4 X5 W X7 
where X9 is A, C, E, G, I, L, M, P, R, Q, S, T, or V; and X8 is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, X9 is A or I; and X8 is D, E, or K.
Particularly preferred peptides include: G G C A D G P T L R E W I S F C G G; G N A D G P T L R Q W L E G R R P K N; G G C A D G P T L R E W I S F C G G K; T I K G P T L R Q W L K S R E H T S; S I E G P T L R E W L T S R T P H S; L A I E G P T L R Q W L H G N G R D T; C A D G P T L R E W I S F C; and I E G P T L R Q W L A A R A.
In further embodiments of the invention, preferred peptides for use in this invention include peptides having a core structure comprising a sequence of amino acids:
C X2 X3 X4 X5 X6 X7 
where X2 is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, L, M, R, S or V; X4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, S, V or Y; X6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V. In a more preferred embodiment, X4 is A, E, G, H, K, L, M, P, Q, R, S, T, or W. In a further embodiment, X2 is S or T; X3 is L or R; X4 is R; X5 is D, E, or G; X6 is F, L, or W; and X7 is I, K, L, R, or V. Particularly preferred peptides include: G G C T L R E W L H G G F C G G.
In a further embodiment, preferred peptides for use in this invention include peptides having a structure comprising a sequence of amino acids:
X8 C X2 X3 X4 X5 X6 X7 
where X2 is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, L, M, R, S, V or W; X4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W or Y; X7 is C, G, I, K, L, M, N, R or V; and X8 is any of the 20 genetically coded L-amino acids. In some embodiments, X8 is preferably G, S, Y, or R.
In another embodiment, the peptide compounds of the present invention are preferably dimerized or oligomerized to increase the affinity and/or activity of the compounds. Examples of preferred dimerized peptide compounds include, but are not limited to, the following: 
In yet a further embodiment, preferred peptides for use in this invention include peptides that are covalently attached to one or more of a variety of hydrophilic polymers. Suitable hydrophilic polymers include, but are not limited to, polyalkylethers as exemplified by polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran and dextran derivatives, etc. It has surprisingly been discovered that when the peptide compounds are derivatized with such polymers, their solubility and circulation half-lives are increased with little, if any, diminishment in their binding activity. In a presently preferred embodiment, the peptide compounds of this invention are dimerized and each of the dimeric subunits is covalently attached to a hydrophilic polymer. In a further preferred embodiment, the peptide compounds of this invention are PEGylated, i.e., covalently attached to polyethylene glycol (PEG). Examples of preferred PEGylated, dimerized peptide compositions of this invention include, but are not limited to, the following: 
wherein xe2x80x9cnxe2x80x9d is an interger having a value ranging from about 5 to about 1000, more preferably from about 10 to about 600 and, even more preferably, from about 110 to about 450.
The compounds described herein are useful for the prevention and treatment of diseases mediated by TPO, and particularly for treating hematological disorders, including but not limited to, thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions. Thus, the present invention also provides a method for treating wherein a patient having a disorder that is susceptible to treatment with a TPO agonist receives, or is administered, a therapeutically effective dose or amount of a compound of the present invention.
The invention also provides for pharmaceutical compositions comprising one or more of the compounds described herein and a physiologically acceptable carrier. These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as inhalable powders and solutions and injectable and infusible solutions.